Somatic hypermutation (SHM) generates point mutations in immunoglobulin (Ig) genes and allows for the production of high affinity antibodies. The reaction is important for protection against infection and for the efficacy of vaccines. SHM is initiated by the activation induced deaminase (AID), which deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase II (Pol II). While AID and SHM act preferentially on Ig genes, they also affect numerous non-Ig loci, and the resulting genetic instability contributes to the development of a range of B cell malignancies. The rules that govern AID/SHM targeting in the genome are not well understood. The central objectives of our proposed experiments are to determine the mechanisms responsible for the preferential targeting of AID/SHM to Ig genes and to establish the rules that govern their mis-targeting to other regions of the genome. We will use complementary biochemical, molecular, genetic, and genomic approaches to achieve the following aims: Aim 1. Determine the protein factors that mediate preferential targeting of SHM to Ig genes and determine their mechanism of action. We have identified the DNA sequences responsible for targeting of AID/SHM to Ig genes, and refer to them as DIVAC (diversification activator). The identity of the critical protein factors that bind DIVAC and the mechanism(s) by which they mediate SHM targeting are not known. We will use biochemical methods to identify DIVAC-binding factors and will test their function using gene targeting and powerful SHM reporter assays. We will systematically determine the DNA sequences and protein domains required for SHM targeting and use this information to reconstitute properly targeted SHM in non-lymphoid cells. We will also determine the distinctive epigenetic, transcriptional, and molecular features of a highly mutating target gene so as to test the model, supported by our preliminary data, that DIVAC functions by causing the arrest of Pol II in the mutation target region, thereby creating an optimal substrate for the action of AID. Aim 2. Map the AID/SHM-susceptible regions of the human genome in normal and DNA repair- deficient cells. Using novel lentiviral SHM reporter vectors and high-throughput mapping of proviral integration sites, we will determine: i) the regions of the human genome that are susceptible or resistant to SHM; ii) where in the genome the action of AID is opposed by high-fidelity DNA repair, and iii) how AID/SHM targeting rules are influenced by DIVAC-binding factors and the cell cycle. These experiments will yield AID/SHM vulnerability maps of the human genome that are likely to have important implications for understanding genomic instability in B cell tumors. Together, our proposed studies have a dual significance, both for basic mechanisms of antibody gene diversification and for the causes of cancer.